Infection of individuals by the hepatitis C virus (HCV) is a public health problem for which there is neither an effective treatment nor a vaccine. Anti-HCV protease drug development programs are in place in several pharmaceutical firms. Like the HIV virus, the HCV genome has a high mutation rate and it is expected that the development of resistance to HCV protease inhibitors will prove to be a roadblock to their efficacy. A clear need exists for the development of procedures capable of prospectively assessing protease drug resistance profiles. Steps in the development of such a procedure are outlined and methods for their implementation described. The procedure will depend upon bacterial expression of a precursor polypeptide such that active HCV NS3 protease is a prerequisite for the expression of a detectable phenotypic marker. Extensive mutation of the protease coding sequence and growth of host bacteria containing the resultant plasmid in the presence of individual or combinations of anti-HCV protease compounds can be used to phenotypically select clones coding for drug resistant proteases. DNA sequence analysis will be used to characterize the spectrum and frequency of mutations contributing to drug resistance. The assay will be used for screening selected anti-HCV protease compounds. PROPOSED COMMERCIAL APPLICATIONS: This work will lead to the development of a system for predicting the spectrum of resistance conferring mutations in the hepatitis C NS3 protease that are selected by therapy using a single anti-protease drug or combination of such drugs. Integration of this system into an anti-HCV protease drug development program will allow the pattern and frequency of resistance conferring mutations to be included as a criteria for clinical lead candidate selection. Compounds can be preselected for optimum resistance conferring properties rather than facing the situation as it stands now where this data is generated in patient populations after the drug development process is completed.